In general, chemical and physical denaturation of protein drugs can be easily caused by unfavorable temperature, shear stress, vibration, freeze-thawing, UV exposure, excessive pH change, organic solvents, microbial contamination, etc. Chemical denaturation includes dimer dissociation, oxidation, deamidation, isomerization, and polymerization, which are influenced by the amino acids constituting the protein and conditions of the solvent containing the protein (salt, pH and temperature). Physical denaturation includes loss of tertiary structure, covalent/non-covalent aggregation and adhesion of monomers, which are influenced by hydrophobic patches on the protein surface changed by protein-containing surrounding environments such as solvents, complex protein structures such as charge distribution, and thermal stability.
The physical or chemical denaturation of a protein including an antibody causes loss of its physiological activities. Since the denaturation is an irreversible process, proteins, once denatured, may not recover their native properties, leading to a reduction in their therapeutic efficacies. It has been also suggested that phenomenon such as aggregation of monomers causes immune responses. Therefore, many studies have been conducted on formulations containing a physiologically effective amount of protein without aggregates (Ishikawa et al., Biol. Pharm. Bull., 33(8): 1413-1417, 2010).
There are many methods available for preventing protein denaturation in liquid formulations. In some protein drugs, the stability problems are addressed via lyophilization. However, the lyophilization process causes stress related to freezing and drying, such as formation of ice crystals, pH change, and high concentration of solute, and these stresses may cause protein denaturation. In addition, since a large-capacity lyophilizer is needed for the lyophilization process during production, high production costs arise during large scale production. Dissolving the lyophilized product in sterile aqueous media before use also poses an inconvenience.
As an alternative to solve these limitations, a stabilizer is added to liquid formulations for the improvement of protein stability. Surfactants, serum albumins, polysaccharides, amino acids, polymers, salts or the like are known as stabilizers used in protein drugs (Wang, Int. J. Pharm., 185: 129-188, 1999; Wang et al., J. Pharm. Sci., 96(1): 1-26, 2007).
In order to prepare stable formulations of the drugs, however, appropriate stabilizers should be used considering the physicochemical properties of each active ingredient. When the stabilizers are used in combination, competition therebetween and adverse effects may lead to undesirable effects. In addition, the concentrations of proteins should be within the range suitable for the stabilization, and in particular, to prepare high concentrations of protein drugs, much effort and caution is required to stabilize proteins in solutions (Shire et al., J. Pharm. Sci., 93(6): 1390-1402, 2004).
Meanwhile, etanercept is a biological modulator that functions as a competitive inhibitor of TNF-α, binding to cell surface TNF-α receptor, to inhibit TNF-α mediated immune responses. Etanercept is a macromolecule with a molecular weight of 150 kDa, and is a homodimer of two fusion proteins linked by disulfide bond, each fusion protein consisting of a human soluble p75 TNF (tumor necrosis factor) receptor coupled to the Fc portion of human immunoglobulin G subclass 1 (Goldenberg, Clinical Therapeutics, 21(1): 75-87, 1999; Moreland et al., Ann. Intern. Med., 130(6): 478-486, 1999).
This is marketed by Amgen under the trade name of Enbrel in 2002. Etanercept is a TNF-α inhibitor used to treat rheumatoid arthritis, psoriasis, and ankylosing spondylitis, and is under clinical trials for the treatment of vasculitis, Alzheimer's disease, and Crohn's disease.
Formulation stabilization technologies for TNFR-Fc fusion proteins such as etanercept aim to develop a liquid formulation for minimizing protein denaturation which may occur during production, storage, and transportation, and maintaining their activity for a long period of time to be identical to that of conventional protein drugs, but it has been difficult to develop satisfactory liquid formulations. Therefore, there is an urgent need to develop a new liquid formulation which is able to stably maintain the activity of TNFR-Fc fusion protein (etanercept) for a long period of time and which is more effective in stabilization of TNFR-Fc fusion protein (etanercept) than the known formulations, including arginine as disclosed in U.S. Pat. No. 7,648,702.